This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

Este protocolo sigue las directrices LUMC del comité de ética de la investigación animal. 1. Preparación de instrumentos, medio de cultivo, y MTCS Nota: Realice todas las preparaciones en la campana de flujo laminar. La cámara de perfusión MTCS, trampa de burbujas y el soporte se describen en Lieber et al., 2010 11. <ol><li> Desinfectar los fórceps y micro tijeras con etanol al 70%. Preparar una jeringa de 5 ml con una aguja 21G con NaCl estéril de Tyrode Buffer (130 mM, 5,4 mM KCl, mM Hepes 6,0, 1,2 mM MgSO 4, 1,2 mM KH 2 PO 4, mM glucosa 20, 1,5 mM CaCl 2 • 2H 2 O, pH = 7,2). Preparar una jeringa de 5 ml con una aguja 21G con una solución de KCl estéril (100 mM). </li><li> Preparar 65 ml de medio por DMEM: corazón suplementado con 10% suero de ternero fetal, de antibiótico / antimicótico (A / A; 100 unidades de penicilina, 100 g de estreptomicina y 0,25 ug de anfotericina B / ml), insulina-Transferrin-El selenio (ITS; 10 mg / ml de insulina, 5,5 mg / ml de transferrina, 6,7 ng / ml selenita de sodio) y esterilizar filtro. </li><li> Esterilizar la cámara de perfusión y la trampa de burbujas mediante pulverización con etanol al 70% y secar con una toalla de papel. Montar el MTCS (Figura 1D), sin embargo, inicialmente sin la cámara de perfusión. Coloque la trampa de burbujas en el estrado. Utilice una bomba de rodillo peristáltico con cabezales de bomba fácil de carga. <ol><li> Uso de tubo de silicona hacer conexiones entre el depósito (tubo de 50 ml) y la bomba, la bomba y la trampa de burbujas, la trampa de burbujas y el depósito (véase la Figura 1D). Hacer el tubo dentro de la incubadora tiempo suficiente para permitir medio alcance el equilibrio de gas con el gas en la incubadora a través de la tubería de gas permeable de silicio (1 m) y el tubo fuera de la incubadora tan corto como sea posible. </li></ol></li><li> Llene el tubo de 50 ml con etanol al 70%, encienda la bomba (velocidad de flujo alrededor de 1 ml / min para permitir la circulación adecuada) y dejar que el cir etanolcular durante 30 min para esterilizar todos los tubos. Separar el etanol mediante el vaciado del depósito y bombear el etanol a partir del sistema. </li><li> Llene el tubo de 50 ml con agua destilada estéril, encender la bomba y deje circular el agua durante 30 minutos para deshacerse de etanol restante. Eliminar el agua vaciando el depósito y bombear el agua desde el sistema. </li><li> Llene el tubo de 50 ml con 45 ml de medio, poner el pie en la incubadora. Encienda el (velocidad de flujo alrededor de 1 ml / min) la bomba y circule el medio para llenar toda la tubería, retire todas las burbujas de aire y permitir que el medio para adaptarse a la composición del gas en la incubadora durante al menos 1 hora. Mantener la incubadora a condiciones de cultivo de tejidos estándar (5% de CO 2 y 37 ° C). Asegúrese de que no hay burbujas en el tubo. </li></ol> 2. Aislamiento de ratón del corazón <ol><li> Inyectar el ratón con 500 unidades de heparina. Esto evitará que la sangre se coagule y permite la eliminación de blood desde el corazón. Después de 10 min, anestesiar el ratón en una cámara de inducción con 4% de isoflurano usando un vaporizador de precisión. La anestesia es suficiente cuando el ratón no responde a la pizca dedo del pie. </li><li> Transferir el ratón para un tablero de disección y mantener la anestesia por medio de una mascarilla conectada a un circuito coaxial. Desinfectar la piel del ratón con un 70% de alcohol. Usando tijeras abiertas de la cavidad abdominal para exponer la vena cava y el diafragma. </li><li> Para exponer el corazón, hacer incisiones laterales a partir de la última a las primeras costillas y reflejan la caja torácica sobre la cabeza de ratón. Detener la anestesia como el ratón no está respirando más. Observar los latidos del corazón. </li><li> Insertar la aguja 21G unida a una jeringa 5 ml que contiene tampón estéril de Tyrode en la vena cava inferior desde el abdominal en la cavidad torácica (a través de diafragma). Asegúrese de que la sangre pueda salir de la vena cava caudal de la inserción de la aguja. Perfundir ªBuffer de correo Tyrode con suavidad y con una presión constante en la vena cava hasta que el corazón pierde en parte su color rojo. Nota: El corazón permanece superando lo que permite la extracción de la sangre desde el corazón. </li><li> Vuelva a colocar la aguja con la aguja 21G unida a una jeringa de 5 ml que contiene la solución KCl estéril. Perfundir suavemente la solución de KCl en la vena cava hasta que el corazón deja de latir y retire la aguja. Nota: La solución de KCl conserva el corazón en la fase diastólica relajado, lo que permitirá la inserción más fácil de las agujas de perfusión y la perfusión del sistema coronario. </li><li> Levante ligeramente el corazón utilizando pinzas curvas y con unas tijeras diseccionar el corazón libre de los tejidos circundantes, pero dejan las arterias y las venas proximales al corazón intacto y unido al corazón. Transferir el corazón a un tubo de 15 ml que contiene PBS enfriado con hielo suplementado con antibiótico / antimicótico (A / A). Guarde el corazón helado de PBS durante un máximo de 3 horas. </li></ol> 3. Cannulation de los corazones del ratón en la cámara de perfusión <ol><li> Realizar todos los procedimientos en campana de flujo laminar. Transferir el corazón aislado del tubo de 15 ml a una placa de 10 cm Petri y añadir PBS suplementado con A / A. </li><li> Bajo un microscopio de disección, utilice la tijera micro y fórceps para eliminar todo el tejido no cardiaco pero preservar la aorta ascendente al menos a la bifurcación con la arteria braquiocefálica y las venas pulmonares, 2 mm proximal al corazón. Cortar la punta de la punta del corazón para crear el acceso al lumen del ventrículo izquierdo (típicamente 2 mm). Coloque la cámara de perfusión en el campana de flujo bajo el microscopio de disección. </li><li> Adjuntar una jeringa de 5 ml con medio a la aguja de inserción usando el tubo de silicona. Llenar la cámara de perfusión con 20 ml de medio y poner el corazón en la etapa de rotación entre las 2 agujas romas en la cámara de perfusión (Figura 1). Ajuste la altura de la etapa de rotación por lo que el corazón se coloca delante de laagujas. </li></ol> 4. La ligadura para el cultivo de la válvula mitral (ver Figura 1) <ol><li> Ligar la aorta con sutura (seda 7.0). Ligar la orejuela auricular izquierda con sutura. Insertar la aguja (nr. 1 en la Figura 1) a través de la vena pulmonar hacia la aurícula izquierda y se liga con sutura proximal de su entrada en la aurícula izquierda. Asegúrese de que la aguja no está demasiado lejos en la aurícula izquierda ya que esto podría dañar la válvula mitral. </li><li> Insertar la aguja (nr.2 en la Figura 1) con movimiento lineal en el ventrículo izquierdo. Transferir el medio procedente de la cámara de perfusión a un tubo de 50 ml. Sellar la aguja al miocardio con pegamento biocompatible. </li><li> Después de que el pegamento se haya secado, inyecte cuidadosamente medio en el corazón mediante la conexión de una jeringa llena de mediano a la aguja nr.1 y compruebe si hay alguna fuga. Asegúrese de que las salidas de medianas de nr.2 aguja y la última sangre restante se perfunde fuera del corazón. </li></ol> 5. Ligación para el cultivo de la válvula aórtica (ver Figura 1) <ol><li> Insertar la aguja (Nr.1 en la Figura 1) en la aorta y se liga con sutura. Asegúrese de que la aguja no está demasiado lejos hacia la aorta ya que esto podría dañar la válvula aórtica. Insertar la aguja (nr.2 en la Figura 1) con movimiento lineal en el ventrículo izquierdo. Transferir el medio procedente de la cámara de perfusión a un tubo de 50 ml. Sellar la aguja al miocardio con pegamento biocompatible. </li><li> Después de que el pegamento se haya secado, inyecte cuidadosamente medio hasta el corazón mediante la conexión de una jeringa llena de mediano a la aguja nr.2 y compruebe si hay alguna fuga. Asegúrese de que las salidas de medianas de nr.1 aguja y la última sangre restante se perfunde fuera del corazón. </li></ol> 6. Coloque cámara de perfusión en el Stand <ol><li> Llenar cámara de perfusión con los 20 ml de medio transferidos. Coloque la junta y la tapa de la cámara y apretar con lavadora y tornillos. </li><li> Retire los MTCS reunidos desde el INCUBAtor. Coloque la cámara de perfusión en el estrado. Conecte la tubería (ver Figura 1D). Coloque el soporte en la incubadora (5% de CO 2 y 37 ° C). Conectar el tubo a la bomba. Encienda la bomba con velocidad de flujo alrededor de 600 l / min. </li><li> Asegúrese de que en el depósito y / o la trampa de burbujas el nivel del medio permite que las visualizaciones de la presencia del flujo por la caída de las gotas medianas entrantes. Después del cultivo, el corazón es retirado del sistema, fijaron y procesaron para examen histológico. </li></ol>

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	<li>Go, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2013).</li><li>Gould, S. T., Srigunapalan, S., Simmons, C. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease.">Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease.</a> Circulation Research. 113 (2), 186-197 (2013).</li><li>Kruithof, B. P. T., Krawitz, S. A., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling.">Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling.</a> Developmental biology. 302 (1), 208-217 (2007).</li><li>Schoen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+valves+and+valvular+pathology:+update+on+function,+disease,+repair,+and+replacement.">Cardiac valves and valvular pathology: update on function, disease, repair, and replacement.</a> Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology. 14 (4), 189-194 (2005).</li><li>Balachandran, K., Sucosky, P., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification.">Hemodynamics and mechanobiology of aortic valve inflammation and calcification.</a> International journal of inflammation. , 263870 (2011).</li><li>Butcher, J. T., Simmons, C. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+the+Aortic+Heart+Valve.">Mechanobiology of the Aortic Heart Valve.</a> The Journal of heart valve disease. 17 (1), 62-73 (2008).</li><li>Balachandran, K., Konduri, S., Sucosky, P., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch.">An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch.</a> Annals of biomedical engineering. 34 (11), 1655-1665 (2006).</li><li>Weiler, M., Hwai Yap, C., Balachandran, K., Padala, M., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets.">Regional analysis of dynamic deformation characteristics of native aortic valve leaflets.</a> Journal of Biomechanics. 44, 1459-1465 (2011).</li><li>Combs, M. D., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+Regulatory+networks+in+development+and+disease.">Heart valve development: Regulatory networks in development and disease.</a> Circulation Research. 105, 408-421 (2009).</li><li>Kruithof, B. P. T., Duim, S. N., Moerkamp, A. T., Goumans, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+and+BMP+signaling+in+cardiac+cushion+formation:+lessons+from+mice+and+chicken.">TGF&#946; and BMP signaling in cardiac cushion formation: lessons from mice and chicken.</a> Differentiation; research in biological diversity. 84 (1), 89-102 (2012).</li><li>Lieber, S. C., Kruithof, B. P. T., Aubry, N., Vatner, S. F., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves.">Design of a miniature tissue culture system to culture mouse heart valves.</a> Annals of biomedical engineering. 38 (3), 674-682 (2010).</li></ol>

The authors declare no conflicts of interest.

Los pasos críticos en el cultivo de las válvulas de ratón cardíacas incluyen hacer el tiempo entre la extirpación del corazón desde el ratón y la ligadura en la cámara de perfusión tan corto como sea posible para asegurar la viabilidad y la ligadura de las agujas perpendicular a las válvulas para garantizar la dirección apropiada de la corriente de . Adicionalmente, control del caudal después de la ligadura en la cámara de perfusión sin medio asegura la inserción y la ligación de agujas apropiada. Es fun...

Enfermedad de las válvulas cardíacas son una causa importante de morbilidad y mortalidad en el mundo occidental; su prevalencia aumenta con la edad y afecta a más del 10% de la población de 75 años y mayores 1. Las válvulas de la parte sistémica del corazón, las válvulas aórtica y mitral, son en su mayoría afectada. Enfermedad de la válvula cardíaca se caracteriza por la pérdida de la estructura altamente organizada de las válvulas, lo que resulta en la alteración de las propiedades mecánicas 2. La integridad estructural tanto, es fundamental para la función de la válvula. Las valvas de la válvula se componen de células intersticiales valvulares (VIC), células endoteliales valvulares (VEC), y la matriz extracelular, que está altamente organizado en un patrón en capas 3,4. Los VIC son responsables de la síntesis de ECM, la degradación y la organización. Factores que emana de la circulación sanguínea, EC o resida en el acto de ECM en los VIC orquestar su función. En adición,fuerzas mecánicas actúan sobre la valva durante el ciclo cardíaco que resulta en laminar o tensión de cizallamiento oscilatorio, tensiones de compresión que influyen en la resistencia a la tracción o comportamiento de VIC 5. Para entender cómo se regula la estructura de la válvula, primero hay que entender cómo VIC responden a la diversa serie de estímulos experimentados durante el ciclo cardíaco. Los estudios in vitro han sido muy informativo sobre las características y capacidades de las células valvulares. La respuesta de estas células in vitro, sin embargo, no siempre puede imitar con precisión la respuesta in vivo 6; por ejemplo, la respuesta de VIC a los estímulos depende de la presencia de ECs y la composición ECM 5. Además, la respuesta de las células a los estímulos valvulares depende de su ubicación específica en el prospecto 7. Además de los estímulos bioquímicos, el comportamiento de las células valvulares se determina por las fuerzas mecánicas que actúan on la válvula 8. Cada región de la válvula se somete a su propio conjunto específico de tensiones hemodinámicas. Aunque los modelos actuales ex vivo han demostrado que las fuerzas mecánicas son determinantes importantes de la estructura valvular 5, los mecanismos asociados aún no están claros. Mientras que los modelos in vivo han proporcionado información sobre los mecanismos moleculares que subyacen el desarrollo valvular 9,10, conocimientos sobre la biología valvular adultos sigue siendo difícil de alcanzar. Por lo tanto, un modelo in vivo ex flujo fue desarrollado en el que las válvulas cardíacas pueden cultivarse en su posición natural en el corazón durante un período prolongado de tiempo 11. Esto tiene la ventaja de que las válvulas se mantienen en su configuración natural y los VIC experimentan el mismo entorno que in vivo, por lo que las respuestas a los estímulos vics tan natural como sea posible. Además, la cultura de las válvulas en su posición natural en el corazón facilita sometiendo cada unoregión valvular a las tensiones hemodinámicas relevantes. En este modelo ex vivo, es decir, el sistema de tejido miniatura Cultura (MTCS), las válvulas se puede someter a diferentes estímulos bioquímicos y hemodinámicos que permiten la investigación de su papel en la remodelación de la válvula cardiaca.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>life technolgies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>life technolgies</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>life technolgies</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>life technolgies</td>
			<td>15240-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk 7-0</td>
			<td>Ethicon</td>
			<td>768G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dish</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe</td>
			<td>BD</td>
			<td>309649</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>BD</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>Tedpella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>Thermo Scientific</td>
			<td>8060-0020</td>
			<td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td>
		</tr>
		<tr>
			<td>Silicon tubing for pump</td>
			<td>Masterflex</td>
			<td>96400-13</td>
			<td>I.D. x O.D. x Wall: 0,8 x1,59 x 0,40 mm</td>
		</tr>
		<tr>
			<td>Biocompatible glue (Histoacryl)</td>
			<td>B. Braun</td>
			<td>1050071</td>
			<td></td>
		</tr>
		<tr>
			<td>precision vaporizer</td>
			<td>Dr&#228;ger</td>
			<td>Vapor 200</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic roller pump</td>
			<td>Masterflex</td>
			<td>7521-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-load pump head</td>
			<td>Masterflex</td>
			<td>7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow chamber</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
	</tbody>
</table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

La válvula aórtica (Figura 2) o la válvula mitral 11 pueden ser cultivadas durante al menos 3 días. Mediante el cultivo en la posición abierta (que representa la posición sistólica de la válvula aórtica y la posición diastólica para la válvula mitral), las células permanecen viables valvulares. No se observa la muerte celular tal como se determina por la ausencia de células TUNEL positivas (Figura 2H, I) o exfoliados caspasa-3 de expresión (que no se muestra y ...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

El cultivo de ratón cardíaca válvulas en el sistema de cultivo tisular miniatura

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

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JoVE Journal

Bioengineering

8.7K vistas.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Video: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Un método para la encapsulación de los folículos ováricos y la Cultura en un proteolíticamente degradables tres dimensiones del sistema

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Investigación

Bioingeniería

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

La encapsulación de los cardiomiocitos en un hidrogel de fibrina para ingeniería de tejidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Fuerza Litografía Cardiaca de Ingeniería de Tejidos

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Las reconstrucciones anatómicas del sistema venoso cardíaco humana usando tomografía computarizada con contraste de las muestras de perfusión fijos

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocolo para la evaluación relativa hidrodinámico de Tri-lámina polimérica Válvulas

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingeniería de tejidos de un humano 3D<em&gt; In vitro</em&gt; Sistema de Prueba de Tumores

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Órgano Cultura Modelo Murino del disco intervertebral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamífero en andamios tridimensionales péptido

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

Un método basado en el molde neto creación libre de andamio tridimensional de tejido cardiaco

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxiales caracterizaciones mecánicas de válvulas auriculoventricular del corazón

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...